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Environmental Pollution 213 (2016) 600e607

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Assessment of status of three water bodies in Serbia based on tissue

metal and metalloid concentration (ICP-OES) and genotoxicity (comet
Karolina Sunjog a, b, *, Stoimir Kolarevi
c b, Margareta Kracun-Kolarevi
c c,

Zeljka Visnji c , Stefan Skori
c-Jefti a
c , Zoran Ga
c , Mirjana Lenhardt a, Nebojsa Vasi
c d,
Branka Vukovi c-Gacic b

Department of Natural Resources and Environmental Sciences, Institute for Multidisciplinary Research, Kneza Viseslava 1, University of Belgrade, 11000
Belgrade, Serbia
Center for Genotoxicology and Ecogenotoxicology, Chair of Microbiology, Faculty of Biology, Studenski Trg 16, University of Belgrade, 11000 Belgrade,
Institute for Biological Research Sinisa Stankovic, Despota Stefana 142, University of Belgrade, 11000 Belgrade, Serbia
Department of Mineralogy, Crystallography, Petrology and Geochemistry, Faculty of Mining and Geology, University of Belgrade, Djusina 7, 11000 Belgrade,

a r t i c l e i n f o a b s t r a c t

Article history: Metals and metalloids are natural components of the biosphere, which are not produced per se by
Received 26 January 2016 human beings, but whose form and distribution can be affected by human activities. Like all substances,
Received in revised form they are a contaminant if present in excess compared to background levels and/or in a form that would
1 March 2016
not normally occur in the environment. Samples of liver, gills, gonads and muscle from European chub,
Accepted 2 March 2016
Squalius cephalus, were analyzed for Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn using inductively
coupled plasma optical emission spectrometry (ICP-OES) to highlight the importance of tissue selection
in monitoring research. The comet assay or single cell gel electrophoresis (SCGE) was selected as an
in vivo genotoxicity assay, a rapid and sensitive method for measuring genotoxic effects in blood, liver
Comet assay and gills of the European chub. Microscopic images of comets were scored using Comet IV Computer
Genotoxicology Software (Perceptive Instruments, UK).
Fish The objective of our study was to investigate two reservoirs, Zlatar and Garasi, and one river, Pestan by:
Metals (i) determining and comparing metal and metalloid concentrations in sediment, water and tissues of
Metalloids European chub: liver, gills, muscle and gonads (ii) comparing these ndings with genotoxicity of water
expressed through DNA damage of sh tissues.
A clear link between the level of metals in water, sediment and tissues and between metal and
genotoxicity levels at examined sites was not found. This suggests that other xenobiotics (possibly the
organic compounds), contribute to DNA damage.
2016 Elsevier Ltd. All rights reserved.

1. Introduction

Metals are one of the serious pollutants in our natural envi-

ronment due to their toxicity, persistence and bioaccumulation
* problems (Tam and Wong, 2000; Torre et al., 2013; Aliko et al.,
This paper has been recommended for acceptance by Maria Cristina Fossi.
* Corresponding author. Center for Genotoxicology and Ecogenotoxicology, Chair 2015). Although metals are naturally occurring elements that are
of Microbiology, Faculty of Biology, Institute for Multidisciplinary Research, Uni- found throughout the Earth's crust, most environmental contami-
versity of Belgrade, 11000 Belgrade, Serbia. nation and human exposure result from anthropogenic activities
E-mail addresses: sunjogkarolina@imsi.rs, sunjogkarolina@yahoo.com such as mining and smelting operations, industrial production and
(K. Sunjog), stoimirk@bio.bg.ac.rs (S. Kolarevi c), margareta.kracun@ibiss.bg.ac.rs
(M. Kracun-Kolarevi c), nebojsa.vasic@rgf.bg.ac.rs (N. Vasi
c), brankavg@bio.bg.ac.rs
use, and domestic and agricultural use of metals and metal-
(B. Vukovic-Gaci
c). containing compounds. Because of their high degree of toxicity,

0269-7491/ 2016 Elsevier Ltd. All rights reserved.
K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607 601

arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), and mercury the diabase-chert formation led to increased content of chromium
(Hg) rank among the priority metallic elements that are of public and nickel within the recent sediments.
health signicance. These metal and metalloid elements are Reservoir Garasi was chosen as the site with low anthropogenic
considered systemic toxicants that are known to induce multiple impact and it is used as a drinking water source. Recent sediments
organ damage, even at lower levels of exposure (Tchounwou et al., at reservoir Garasi are feeding from three geological complexes:
2012). It has been reported that metals such as cobalt (Co), copper Bukulja granitoid, thermo-contact metamorphosed Upper Creta-
(Cu), chromium (Cr), iron (Fe), magnesium (Mg), manganese (Mn), ceous sedimentary rocks (phyllite, ne-grained gneisses,
molybdenum (Mo), nickel (Ni), selenium (Se) and zinc (Zn) that are muscovite-biotite schists and marbles) and Miocene sediments
required as essential nutrients for various biochemical and physi- from the Belanovica Lake basin. Each of them carries its own
ological functions (WHO, 1996a) can become toxic when environ- petrology and geochemical record.
mental concentrations are increased. However, because they are River Pestan, is a polluted site at Kolubara basin, which has rich
natural, they have certain unique properties that distinguish them deposits of lignite (brown coal), and hence, the whole area is under
from human-produced contaminants as well as from organic sub- intensive mining activity. Geological resources that nourish Pestan
stances in general (Chapman and Wang, 2000). are petrologically highly heterogeneous. Right bank is from Plio-
The sediments are polluted with various kinds of hazardous and cene and Early Quaternary deposits. Left bank is much more com-
toxic substances, including metals. In some conditions, more than plex. It consists of Paleozoic rocks, Miocene sediments, acid
99% of metals entering the river stream can be accumulated in volcanics and the Miocene granitoid Brajkovac. Elevated values of
sediments in various forms (Salomons, 1995, Peng et al., 2009). chromium and nickel in Pestan are the consequence of supply from
However, metals cannot stay xed in sediment forever. With the Cretaceous clastic rocks, which have been signicantly fed from
variation of the physicalechemical characteristics of water condi- ultrabasic rocks. Ultrabasic rocks are the primary carriers of chro-
tions, part of these xed metals will re-enter the overlying water mium, nickel and cobalt.
and become available to living organisms.
Metals in the sh body are accumulated in different amounts 2.2. Water and sediment sampling
and these differences result from different afnity to tissues,
different uptake, deposition and excretion rates (Jezierska and Information regarding the quality of water and sediment based
Witeska, 2006). The uptake of metals in sh was found to occur on chemical analyses (metals and organic pollutants) was obtained
through absorption across the gill surface, through the gut wall from the Republic Hydrometeorological Service of Serbia.
tract and skin (Obasohan, 2007, Fazio et al., 2014). At reservoirs Garasi water was sampled at 3 depths (0.5, 10,
The Europian chub, as other sh belonging to the family Cyp- 20 m) and 3 locations and sediment was sampled at 3 locations. At
rinidae, offers possibilities to investigate water bodies with various Uvac water was sampled at 3 depths (0.5, 20, 40 m) and 3 locations
ecological status (Durand et al., 1999; Liew et al., 2015; Chromcova and sediment was sampled at 1 location. At Pestan water and
et al., 2015). The use of chub as a bioindicator has already been sediment were sampled at one location and one depth. Sampling
reported in eld studies assessing the environmental quality of trials for water were at Uvac 07/2009, 08/2010, 09/2011, at Garasi
inland waters differently impacted by anthropogenic activities 08/2009, 09/2010, 08/2011, and at Pestan 11/2009, 11/2010, 10/2011.
(Winter et al., 2004; Randak et al., 2006; Kr ca et al., 2007; Pavlica Sampling trials for sediments were at Uvac in 08/2010 and in 09/
et al., 2011; Sunjog et al., 2013, 2014). 2011; at Garasi in 08/2009, 09/2010 and 08/2011, and at Pestan in
The comet assay or single cell gel electrophoresis (SCGE) was 08/2010 and 08/2011.
selected as an in vivo genotoxicity assay, as a rapid and sensitive
method for measuring genotoxic effects in individual cells 2.3. Fish sampling
(Fairbairn et al., 1995). It combines the simplicity of biochemical
techniques for detecting DNA single-strand breaks (strand breaks Field sampling was conducted at three sites located in Serbia,
and incomplete excision repair sites), alkali labile sites, and cross- Pestan (N 44 260 05.2600 E 20 150 22.9500 ), Garasi (N 44170 29.7100 E
linking, with the single-cell approach, typical for cytogenetic as- 20 280 54.7400 ) and Uvac (N 43 290 56.900 E 19 490 27.300 ). Species of
says (Kumaravel et al., 2009). This assay has found its application in European chub analyzed in this study are occurring naturally in
basic research as well as in applied sciences such as medicine, these water bodies. Total of 18 specimens were caught at all sites,
ecogenotoxicology, monitoring studies, etc. (Frenzilli et al., 2009; where at Uvac (7 specimens) and Garasi (5 specimens) were caught
Meybodi and Mozdarani, 2009; Kolarevi c et al., 2011; Sunjog by gillnets (dimensions 10e30 m  1e2 m, 10e80 mm mesh size),
et al., 2014; Ga
cic et al., 2014). and at Pestan (6 specimens) by electro shing device ELEMAX SHX
The objective of our study was to investigate two reservoirs, 2000 (SAWAFUJI). The sampling trails were from October to
Zlatar and Garasi, and one river, Pestan by: (i) determining and November 2011.
comparing metal and metalloid concentrations in sediment, water
and tissues of European chub: liver, gills, muscle and gonads (ii) 2.4. Tissue sample collection and preparation
comparing these ndings with genotoxicity of water expressed
trough DNA damage in tissues of European chub: blood, liver, and Fish are anesthetized with clove oil prior to tissue collection and
gills. dissection. The total mass (g) and the total body length (cm) of each
specimen were measured. For metal analysis samples of liver, gills,
2. Materials and methods muscle, and gonads were quickly removed, washed with distilled
water, and stored at 20  C. For comet assay blood was collected
2.1. Sampling location using a syringe directly from the heart and added to microtubes
with 0.5 mL cooled Heparin-PBS (Phosphate Buffered Saline) so-
Reservoir Zlatar, formed on river Uvac, is a protected natural lution to prevent clotting and liver and gills were removed and
area of great importance with very low anthropogenic impact. The added to microtube with 3 mL cooled PBS. After that liver and gills
land on which now lake sits is of Triassic and Jurassic products, and were excised and chopped separately 10 times in 0.2 ml of HBSS
of less exposed Miocene lacustrine sediments. The supply of ma- (Hank's Balanced Salt Solution) using two fresh scalpel blades in a
terial from basic igneous rocks (spillite, diabase and gabbro) from scissor-like movement on a Petri dish, washed off gently into a
602 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607

15 ml centrifuge tube with a further 2.5 ml HBSS and 0.3 ml of parameters (between tissues and sites) were performed by the
trypsin (nal conc. 0.05%). The incubations were gently shaken for non-parametric ManneWhitney test for two independent samples.
10 min at room temperature, after which 10 ml of HBSS was added Detection of multivariate outliers was performed with Hotel-
and the suspension passed through a sieve to remove any large ling's T2 test.
fragments that remained. After centrifugation (2000 rpm for
10 min), the supernatant was discarded and the pellet carefully 3. Results and discusion
resuspended in 0.5 mL of HBSS.
3.1. Metal, metalloid and organic pollutants analysis in water and
2.5. Analysis of metals and metalloids sediment

Frozen tissue samples of liver, gills, muscle, and gonads were Average values of heavy metal concentrations in water and
rst measured (wet weight) and then dried by Freeze Dryers sediment and organic pollutants in sediment for 2009, 2010, and
Rotational Vacuum Concentrator, GAMMA 1e16 LSC, Germany. 2011year are presented in Table 1. According to data analysis in
After that samples were measured again (rst dry weight) and water Mn and Pb were elevated at Garasi, Zn, Cu, Cr and Cd at Uvac,
sample portions between 0.2 and 0.5 g (second dry weight) were while Fe, Ni and As was higher at Pestan comparing to Garasi and
processed in a microwave digester (speed wave MWS-3; Bergof Uvac (Table 1a). According to National regulative (Ofcial Gazette
Products Instruments GmbH, Eningen, Germany), using 6 mL of 16/2011) all element concentrations are in rst class quality of
65% HNO3 and 4 mL of 30% H2O2 (Merck suprapure) at a food water, except As at Pestan which was in second class.
temperature program (100e170  C). The digested samples were Sediment analysis showed that Cu and Cd were highest at
diluted with distilled water to a total volume of 25 mL, and the Garasi, while Fe, Mn, Cr and Ni were elevated at Uvac and Zn, Pb, Hg
analysis was performed by inductively coupled plasma optical and As were highest at Pestan (Table 1b). At Uvac Cr with 255.5 mg/
spectrometry (ICP-OES). The following 12 metals and metalloids kg and Ni with 421 mg/kg was higher than their MAC (Maximum
were analyzed: Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn. The Allowable Concentration) values for sediment (Cr 240 mg/kg,
following wavelength lines of the ICP-OES analysis were used: Al Ni 44 mg/kg) according to National regulative (Ofcial Gazette
394.401, As 189.042, B 249.773, Ba 233.527, Cr 205.552, Cu 324.754, 16/2011). At Garasi Cu was 188.22 mg/kg which was also above
Fe 259.941, Hg 194.227, Mn 259.373, Mo 202.095, Sr 460.733, and MAC value for sediment (Cu 110 mg/kg).
Zn 206.191. BCR-185R reference material of bovine liver as well as Presented data suggests on relatively uniform distribution of
IAEA-336 Lichen reference material was used for the assessment of chalcophile elements: at Uvac 279.5 ppm, at Garasi 364.4 ppm, and
the accuracy and precision of the analysis. Analysis indicated that at Pestan 325.8 ppm. However, the total concentrations of the
the concentrations were within 90e115% of the certied values for metals such as Cr and Ni in these three localities were found to be
all measured elements. All metal and metalloid concentrations different: at Uvac 676.5 ppm, at Garasi 78.7 ppm and at Pestan
were expressed as mg g1 dry weight (dw). 279.4 ppm. The likely sources of nickel and chromium contami-
nation in recent sediments in the river Uvac are the basic igneous
2.6. Comet assay rocks (spilite, diabase and gabbro) from the diabase-chert forma-
tion. Elevated concentrations for nickel and chromium in Pestan are
The alkaline comet assay procedure was performed by the the consequence of contribution of material from Cretaceous clastic
method described by Singh et al. (1988) with some modications rocks, feeder signicantly from ultrabasic rocks. Ultrabasic rocks
(Sunjog et al., 2014). Microscopic slides were precoated with two are considered the primary carriers of chromium, nickel and cobalt.
layers of 1% NMP (normal melting point) agarose after which 30 ml Benzo[b]uoronathene and benzo[k]uoronathene was detec-
of cell pellet suspension was gently mixed with 70 ml of 1% LMP ted in similar concentration at Garasi and Pestan and elevated at
(low melting point) agarose and pipetted on the supportive layer of Uvac. Fluoronathene was highest at Uvac, than Garasi and the
1% NMP agarose. Slides were then placed into cold lysis buffer for smallest value was detected at Pestan. Alachlor was only elevated at
1 h. To allow DNA unwinding, slides were put into the electro- Garasi and bellow level of detection at Uvac and Pestan. p,p0 DDT
phoresis chamber containing cold alkaline electrophoresis buffer was elevated at Garasi and Uvac and was not detected at Pestan. On
for 20 min. Electrophoresis was performed at 0.6 V/cm at 4  C for the other hand para-tert-octylphenol and methoxychlor was
20 min. After the electrophoresis, slides were placed into freshly elevated at Pestan.
made neutralizing buffer for 15 min. Staining was performed with According to National regulative (Ofcial Gazette 16/2011)
20 ml per slide of EtBr (2 mg mL1). Microscopic images of comets naphthalene, uoronathene and benzo[k]uoronathene are bellow
were scored using Comet IV Computer Software (Perceptive In- MAC, and only DDT (MAC 9 mg/kg) was above at Uvac and Garasi.
struments, UK). Images of 50 cells were captured from each slide
per sample and among the parameters available for analyses the 3.2. Metal analysis in tissues
tail length (tl) tail intensity (ti) and Olive tail moment (tm) were
chosen as parameters to assess the DNA damage, and marked as: Average body length and mass of individuals (used for this an-
blood (btl, bti and btm), liver (ltl, lti and ltm) and gills (gtl, gti and alyses) and average values of heavy metal concentrations are pre-
gtm). sented in Table 2. Inter-group comparisons of the extent of
accumulation among analyzed tissues showed that the differences
2.7. Statistical analysis were statistically signicant for most of the assessed metals and
trace elements (P < 0.05). Most of these inter-site differences for
We have used the principal component analysis (PCA) as an elements are seen in liver and muscle. Gills showed no difference in
unsupervised statistical method that summarize the variation of a accumulation of B, Cr, Fe, Mo i Sr between localities (P < 0.05).
data set between samples to a set of uncorrelated components Gonads were compared only between Garasi and Uvac, because at
(each component is a particular linear combination of the original Pestan concentrations of elements were above detection threshold
variables), in order to assess the differentiation among the analyzed only in two samples, and all elements, except Fe i Hg, showed
sh tissues, based on the element level. signicantly different accumulation. Comparing metal concentra-
Inter-group comparisons of element levels and genotoxicity tion in different tissues showed that gills from Garasi had the
K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607 603

Table 1
Metal and organic pollutants concentrations in water (a) and sediment (b) in 2009/2010/2011 (mean SD) at three studied localities: Garasi, Uvac and Pestan.

a) Water

Metal and metalloids Garasi Uvac Pestan

Fe (mg/l) 0.08 0.09 0.06 0.05 0.38 0.31

Mn (mg/l) 0.31 0.24 <0.01 0.24 0.09
Zn (mg/l) 7.01 7.12 15.04 4.69 5.6 4.19
Cu(mg/l) 5.26 3.2 5.72 1.33 4.9 3.54
Cr(mg/l) 0.38 0.66 0.78 1 0.32 0.39
Pb(mg/l) 1.28 2.21 0.06 0.11 <0.05
Cd(mg/l) 0.33 0.32 0.61 0.76 0.02 0.02
Hg(mg/l) <0.1 <0.01 <0.1
Ni(mg/l) 3.11 2.75 3 0.46 9.95 4.8
As(mg/l) 1.12 0.35 0.75 0.30 6.4 1.27

b) Sediment

Metal and metalloids (mg/kg) Garasi Uvac Pestan

Fe 37,500.11 9275.93 49,521 36,686,11 44,450 19,127,24

Mn 1014.89 611.4 1763 1336.43 782 272.24
Zn 122.89 42.28 120 0 127 9.9
Cu 188.22 61.92 125 74.95 133 77.78
Cr 46.89 14.13 255.5 301.93 134 36.8
Pb 39 26.32 34 16.97 46.5 16.26
Cd 0.99 1.91 0.25 0.35 <0.3
Hg 0.03 0.05 0.1 0 0.25 0.07
Ni 31.78 11.33 421 496.39 145.5 38.9
As 13.22 11.3 <6 19 1.41
Ni Cr 78.7 676.5 279.5
Chalcophile elements* 364.4 279.4 325.8

Organic pollutants (mg/kg)

Benzo(b)uoronathene 4.82 4.69 9.56 9.12 4.35 1.06

Benzo(k)uoronathene 3.31 2.62 5.7 4.38 2.8 0.56
Fluoronathe 9.32 6.62 17.45 15.63 5.3 7.49
Naphthalene 0.46 0.93 <1 0.6 0.85
Para-tert-Octylphenol <2.5 4.15 1.34 7.9 11.17
Alachlor 1.36 4.1 <2 <2
Methoxychlor <1 <1 91.65 129.61
p.p'DDT 20.32 25.17 155.4 87.68 <1

*Chalcophile elements are presented as a sum of Zn, Cu, Pb, Cd, Hg and As.

highest concentration of Al, B, Fe and Mn, at Uvac for As, Mo and Zn, specimens) were within the limits of MAC in sh, given that the
while Ba, Cu, Hg and Sr were highest in gills at Pestan (Fig. 1A, border is 0.5e1 mg/g wet weight, depending on the type of the sh.
Table 2). When analyzing levels in liver we can see that almost all Concentrations of Al, Ba, Cr, Mn, and Sr were highest in gills,
metals were highest at Uvac, except for Al and Hg, which was which made gills the most bioaccumulative tissue. Cu, Fe, and Mo
highest at Garasi and Sr which was above detection level only in were highest in liver, while muscle and gonads had lowest con-
Garasi and Pestan (Fig. 1B, Table 2). For muscle highest levels of Ba, centration in metals from all sites.
Mn and Sr were found at Pestan, and Hg was highest at Garasi while
Al, As, B, Cu, Fe, Mo and Zn were highest at Uvac (Fig. 1C, Table 2). 3.3. Comet assay
For gonads higher concentrations of elements were distributed
between Garasi and Uvac so that Al, As, B, Ba, Fe and Zn were Results of DNA damage measured with comet assay for blood,
highest at Uvac, while Cu, Mn were highest at Garasi (Fig. 1D, liver and gills at all sites for all parameters are presented in Table 3.
Table 2). Inter-group comparisons of genotoxicity parameters showed that
In order to compare heavy metal and trace element concen- Uvac has signicantly lower values of til, tml and tig comparing to
trations in muscles with maximum acceptable concentrations Pestan and Garasi (P < 0.05). Pestan and Garasi differ for blood bti,
(MAC) in sh meat intended for human consumption, as estab- btm, for liver ltl and for gills gtm (P < 0.05). We can see that highest
lished by the European Commission (1881/2006/EC), Food and values for DNA damage were found in gills for all sites, which is in
Agriculture Organization of the United Nations (FAO, 1983), and correspondence with highest heavy metal values in gills. Uvac had
national legislation (Ofcial Gazette, No 32/2002), concentrations the lowest values for all tissues in general, which singled out Uvac
were recalculated to the mg kg1 tissue wet weight (ww). Ac- as a referent site, while Pestan had highest values for all tissues.
cording to regulations of the European Commission, the MACs for
Cd, Pb and Hg in sh are 0.05, 0.3, 0.5e1 mg per gram of wet weight. 4. Discussion
According to regulations of the Republic of Serbia, MAC for As, Zn,
Cu and Fe in fresh meat sh are 2, 100, 30 and 30 mg per gram of wet All metals and metalloids are naturally occurring in the envi-
weight, respectively. MACs for Cu and Zn recommended by FAO ronment, as they are natural components of the Earth's crust.
(1983) amounts to 30 mg per gram of wet weight. Metal concen- Background concentrations of metals and metalloids in the envi-
trations expressed as mg per gram of wet weight in the muscles at ronment are hence above zero and can vary greatly in different
Uvac and Pestan are bellow MAC values for Cd, Pb, Hg, As, Zn, Cu environmental media (e.g., soils, sediments, water) and under
and Fe, while concentration of Hg in muscle at Garasi (in 2 different geological and environmental conditions (Chapman and
604 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607

Table 2
Metal and metalloids concentrations in different tissues of European chub (means standard deviation). Concentrations are expressed as mg g1 dry weight, while ND indicates
the values below the detection threshold.

Garasi Uvac Pestan

TL (cm) 37.72 3.53 22.86 2.14 31.12 5.57

M (g) 643.8 227.65 119.53 25.64 277.37 127.03
Gills Al 146.76 248.29a 1.68 2.15b 22.03 30.47a,b
As 0.40 0.11a 1.72 2.22b 0.16 0.06c
B 0.73 1.24a 0.39 0.31a 0.36 0.14a
Ba 7.13 2.43a 5.75 1.29a 18.55 5.76b
Cr 0.23 0.32a 0.20 0.24a 0.14 0.09a
Cu 1.29 0.66a,b 0.75 0.50b 1.96 0.95a
Fe 255.49 326.31a 108.59 34.39a 90.73 30.15a
Hg 0.93 0.30a ND 1.28 0.27a
Mn 40.64 38.44a 11.24 2.96b 22.66 16.34ab
Mo 0.14 0.10a 0.25 0.38a 0.03 0.03a
Sr 50.66 12.30a 56.20 8.76a 66.42 14.75a
Zn 266.14 43.32a,b 340.02 91.59a 197.67 43.29b
Liver Al 6.22 13.78a 0.21 0.57b 0.11 0.05a
As 0.61 0.32a 1.61 1.22b 0.19 0.09c
B 0.10 0.11a 1.44 0.97b 0.33 0.33a
Ba 0.02 0.00a 0.60 0.55b 0.03 0.02ab
Cr 0.04 0.07a 0.63 0.92a 0.03 0.01a
Cu 10.51 5.81a 31.17 23.57b 18.65 16.62a,b
Fe 44.24 34.36a 495.39 365.87b 128.05 102.69a
Hg 1.35 0.17a ND 1.01 0.28a
Mn 2.07 0.49a 4.19 1.67b 2.60 0.84a
Mo 0.26 0.14a 1.19 0.44b 0.71 0.42b
Sr 0.11 0.05a ND 0.15 0.07a
Zn 29.63 4.87a 113.39 36.31b 51.36 21.34a
Muscle Al 0.08 0.03a 2.26 3.62a 0.09 0.03a
As 0.5 0.28a 1.94 1.05b 0.12 0.05c
B 0.05 0.02a 0.18 0.20b 0.04 0.01a
Ba ND 0.49 0.16a 0.62 0.29a
Cr 0.01 0.01a 0.01 0.02a ND
Cu 1.31 0.62a,b 2.22 0.64a 0.89 0.19b
Fe 7.6 5.23a 21.80 5.69b 7.06 1.74a
Hg 1.95 0.42a 0.78 0.54b 1.78 0.29a
Mn 0.3 0.09a 0.59 0.07b 0.79 0.29b
Mo ND 0.05 0.11a 0.02 0.03a
Sr 0.23 0.07a 1.06 0.51b 1.61 0.42b
Zn 16.62 10.42a 61.09 18.87b 32.57 8.89a
Gonads Al 0.14 0.18a 0.31 0.83b 0.06, 2.05*
As 0.11 0.00a 3.22 0.95b 0.11, 0.55*
B 0.14 0.06a 0.24 0.09b 0.22, 0.13*
Ba 0.02 0.00a 2.26 1.05b 1.14, 2.46*
Cu 5.88 0.95a 1.45 0.55b 5.87, 2.88*
Fe 52.02 17.44a 72.52 34.30a 71.03, 46.41*
Hg 0.90 0.08a 0.72 0.44a 1.05, 1.54*
Mn 25.65 7.20a 0.77 0.38b 17.43, 2.67*
Mo 0.13 0.02a 0.03 0.04b 0.17, 0.15*
Sr 0.47 0.06a 0.42 0.54b 0.47, 0.36*
Zn 96.54 18.64a 365.58 152.21b 231.02, 157.90*
The value with a different letter in the same row is different (Man-Whitney U test, P < 0.05).
*Concentrations above detection threshold only in two samples.

Wang, 2000). Direct correlation of elements in sediment, water and from liver and gills singled out Uvac as the site with the lowest
sh tissues cannot be seen for many reasons: geological composi- genotoxicity compared to Garasi and Pestan. These, seemingly
tion, physical and chemical properties of water, specic accumu- contrary results, may be explained by acclimation/adaptation of
lation pattern in various organs in sh, etc. (Demirak et al., 2006), sh at these fairly constant (without anthropogenic input) levels of
which is also clear from our results. elements in water and geochemical composition of the sediments
As mentioned earlier, Uvac is under state protection for more metal content. This can result in the evolution of resistance, which
than 45 years, without known anthropogenic activities. It could be may have important implications for decisions regarding safe
argued that all metals in water originate solely from sediment, ambient toxicant levels indicating that there can be wide variation
which is again a result of geological composition. According to the in tolerance to toxic agents among different populations of a spe-
sediment analysis Fe, Mn, Cr and Ni were highest at Uvac and water cies (Klerks and Weis, 1987; Chapman and Wang, 2000; Messina
analysis showed that Zn, Cu, Cr and Cd were highest, compared to et al., 2014). There are two reasons why organisms may be resis-
other sites. Analysis of metals and metalloids in tissues from Uvac tant to a pollutant: (1) individual organisms may have acquired a
showed that majority of elements in liver (As, B, Ba, Cr, Cu, Fe, Mn, degree of tolerance by physiological acclimation during exposure to
Mo and Zn) and some in gills (As, Mo, Zn) had higher values than sublethal concentrations at some prior period of their lives. This
samples from other sites. On the other hand, genotoxicity results does not confer tolerance to lethal concentrations upon offspring,
K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607 605

Fig. 1. PCA analysis of metal concentration in gills (a), liver (b), muscle (c), and gonads (d). PCA analysis of metal concentration showed that specimens from Uvac grouped by higher
values for As, Mo and Zn and that from Pestan by higher values for Ba and Sr in gills (a), specimens from Pestan and Garasi by higher values for Al, Hg and Sr and that from Uvac by
higher values for all other analyzed elements in liver (b), specimens from Pestan and Garasi by higher values for Hg and that from Uvac by higher values for Al, As, B, Cu, Fe and Zn in
muscle (c) and specimens from Garasi by higher values for Cu and Mn and that from Uvac by higher values for As, B, Ba, Fe and Zn in gonads (d).

Table 3 Ali, 2008). It is an insecticide preferred to DDT for use on animals,

DNA damage expressed with three parameters, tl, ti and tm, shows the difference in in animal food, and on DDT-sensitive crops such as squash, melons,
level of DNA damage in three tissues (blood, liver and gills) at three water bodies
etc. Since methoxychlor is more unstable than DDT, it has less re-
rivers, Garasi, Uvac and Pestan.
sidual effects. World Health Organization drinking water quality
Parameter Garasi Uvac Pestan standards permit a maximum of 20 mgL1for methoxychlor in water
Blood btl 16.55 4.37a 17.32 4.07a 18.60 4.06a (WHO, 1996b). In sediments from Pestan methoxychlor was found
bti 3.40 3.94ab 3.24 3.49a 5.15 5.06b at a concentration of 91, 65 mg/kg which is potentially hazardous
btm 0.36 0.44ab 0.33 0.38a 0.61 0.61b considering the possibility of re-entering the water and becoming
Liver ltl 19.96 4.75a 22.48 5.05ab 26.49 5.99b
lti 6.77 5.24a 3.74 3.71b 7.02 6.08a
available to living organisms. EPA (1995) has found methoxychlor
ltm 0.82 0.66a 0.49 0.49b 1.08 0.93a to potentially cause central nervous system depression, diarrhea,
Gills gtl 19.88 4.89a 21.87 5.24a 22.01 6.38a and damage to liver, kidney and heart tissue and retard growth
gti 6.79 5.58a 4.34 4.36b 7.36 6.61a from short-term and long-term exposures at levels above the MCL
gtm 0.83 0.70ab 0.56 0.57a 0.93 0.93b
(Maximum Contaminant Level).
The value with a different letter in the same row is different (Man-Whitney U 4-tert-octylphenol is a common environmental pollutant which
test, P < 0.05). acts as an estrogen receptor agonist in sh. Estrogen agonists are
known to interfere with reproduction parameters as well as sexual
development (including changes in sex-ratio) and growth. Expo-
who must also be pre-exposed to acquire it. (2) Populations may
sure to estrogens such as 4-tert-octylphenol may cause long lasting
have evolved genetically based resistance, through the action of
effects. A change in the endocrine feedback system during sensitive
natural selection on genetically based individual variation in
life stages may result in effects during the entire life. In addition to
resistance (Klerks and Weis, 1987). Since the Uvac is a protected
the endocrine disrupting properties, the concern is increased by
area, it is highly likely that the population of sh was adapted to
evidence that 4-tert-octylphenol biodegrades very slowly in the
levels of naturally presented heavy metals and metalloids.
environment and tends to adsorb to sediment and soil. Ferreira-
We cannot say that solely metals and metalloids are responsible
Leach and Hill (2001) reported about biotransformation, bio-
for genotoxicity effects in tissues, but also various mixtures of
concentration and tissue distribution of 4-tert-octylphenol in ju-
organic and other pollution (Sunjog et al., 2012; Bartoskova et al.,
venile rainbow trout (Oncorhynchus mykiss). In a ow-through
2013). Pesticides are considered to be potential chemical muta-
system the sh was exposed with a concentration of 4 m g/l of 4-
gens and potential chronic health hazards. At Pestan methoxychlor
tert-octylphenol for 10 days. The bioconcentration was tissue
(mg/kg) i para-tert-octylphenol (mg/kg) are present in signicant
dependent, where bioconcentration factor in liver and fat were
levels in sediment. Methoxychlor is widely distributed in both
highest. The study suggests that exposure to water-borne
surface water and groundwater throughout the world (Gupta and
606 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607

alkylphenols results in rapid conjugation and elimination of the Fairbairn, D.W., Olive, P.L., O'Neill, K.L., 1995. The comet assay: a comprehensive
review. Mutat. Res. 339 (1), 37e59.
chemical by the liver/bile route, but the high amount of the parent
FAO, 1983. Compilation of Legal Limits for Hazardous Substance in Fish and Fishery
substance can accumulate in a variety of other sh tissues. Products (Food and Agricultural Organization). FAO shery circular, No. 464.
As previously mentioned, heavy metals exhibit different accu- Fazio, F., Piccione, G., Tribulato, K., Ferrantelli, V., Giangrosso, G., Arfuso, F., Faggio, C.,
mulation pattern in different organs. Among different organs, liver 2014. Bioaccumulation of heavy metals in blood and tissue of striped mullet in
two Italian Lakes. J. Aquatic Anim. Health 26 (4), 278e284.
accumulates higher concentrations of metals comparatively and Ferreira-Leach, A.M.R., Hill, E.M., 2001. Bioconcentration and distribution of 4-tert-
has been used widely to investigate the process of bioaccumulation. octylphenol residues in tissues of the rainbow trout (Oncorhynchus mykiss).
Exchange of gases and absorption of heavy metals takes place from Mar. Environ. Res. 51 (1), 75e89.
Frenzilli, G., Nigro, M., Lyons, B.P., 2009. The Comet assay for the evaluation of
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muscles, which exhibit lowest levels of metals accumulation (Jari c to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater
et al., 2011; Akan et al., 2012; Sunjog et al., 2012; Jacimovi
c et al., mussels Unio pictorum and Unio tumidus. Environ. Pollut. 191, 145e150.
2015). Our results conrmed that gills and liver, along with go- Gupta, V.K., Ali, I., 2008. Removal of endosulfan and methoxychlor from water on
carbon slurry. Environ. Sci. Technol. 42 (3), 766e770.
nads accumulate more metals in comparison to muscle. Ja
cimovi c, M., Lenhardt, M., Visnji c-Jefti  Jari
c, Z., c, I., Gacic, Z., Hegedis, A., Krpo-

c, J., 2015. Elemental concentrations in different tissues of European
5. Conclusion perch and black bullhead from Sava Lake (Serbia). Slovenian Vet. Res. 52 (2),
c, I., Visnji c-Jefti  Cvijanovi
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fully appreciated. Specically, environmental concern related to
Jezierska, B., Witeska, M., 2006. The metal uptake and accumulation in sh living in
metals and metalloids should not be triggered only by their pres- polluted waters. In: Twardowska, I., Allen, H.E., H aggblom, M.M., Stefaniak, S.
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Klerks, P.L., Weis, J.S., 1987. Genetic adaptation to heavy metals in aquatic organ-
tration of a metal or metalloid varies greatly in different areas, isms: a review. Environ. Pollut. 45, 173e205.
background or baseline concentrations should be measured, where Kolarevi c, S., Kne zevi c-Vuk cevic, J., Paunovi c, M., Tomovi c, J., Gaci
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in Serbia: microbiological analysis and genotoxicity monitoring. Arch. Biol. Sci.
Our results pointed out the importance of geological back- 63 (4), 1209e1217.
ground of examined sites. A clear link between the level of metals Kr 
ca, S., Zaja,  
R., Cali c, V., Terzic, S., Grubesic, M.S., Ahel, M., Smital, T., 2007. Hepatic
and genotoxicity at examined sites was not found, which suggest biomarker responses to organic contaminants in feral chub (Leuciscus cepha-
lus)-laboratory characterization and eld study in the Sava River, Croatia. En-
that other xenobiotics contribute to DNA damage, possibly the viron. Toxicol. Chem. 26 (12), 2620e2633.
organic compounds. Also it is possible that the same element level Kumaravel, T.S., Vilhar, B., Faux, S.P., Jha, A.N., 2009. Comet assay measurements: a
in tissues does not cause the same stress (in this case DNA damage) perspective. Cell Biol. Toxicol. 25 (1), 53e64.
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Messina, C.M., Faggio, C., Laudicella, V.A., Sanlippo, M., Trischitta, F., Santulli, A.,
2014. Effect of sodium dodecyl sulfate (SDS) on stress response in the Medi-
This study represents a part of activities within the Project No. terranean mussel (Mytilus galloprovincialis): regulatory volume decrease (Rvd)
173045, funded by the Ministry of Education, Science and Tech- and modulation of biochemical markers related to oxidative stress. Aquat.
Toxicol. 157, 94e100.
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